(Adapted from the Applicant's Abstract) Functional mammalian hearts require successful septation and valvulogenesis to form four chambers and a fully differentiated myocardium. The long-term goal is to understand underlying mechanisms for septation, valvulogenesis and differentiation. The investigators previously cloned several novel, differentially-expressed genes (DEGs) including Xin. Initial studies on Xin reveal that it may play important roles in cardiac looping. Incomplete looping has been implicated in septal misalignment/defect in several mouse models. Thus, mouse homologs of these genes together with additional genes to be cloned in Project 2 of this SCOR will be further characterized to understand their roles in cardiac morphogenesis and possibly to reveal their relevance to the etiology of atrial septal, AV canal and ventricular septal defects. Aim 1 is to determine temporal and spatial expression patterns of DEGs. Large-scale, whole-mount in situ screening on DEGs, initially cloned by Project 2 from developing rat hearts, will be carried out. These DEG clones will be prioritized and selected clones will be further characterized. For example, detail in situ hybridization and RNASE protection assay will be used to determine how early the Xin expresses, when its expression reaches maximum and where it is. Simultaneously, full-length mouse clones will be isolated and used for protein and antibody productions. Immunofluorescence will be performed with specific antibody. Aim 2 is to examine the properties of DEG proteins. The predicted protein sequence will be searched for known motifs for which experiments will be designed to test. For example, nuclear localization signal found in Xin has been shown to be functional. Once antibody is available, localization of Xin can be further determined by immunofluorescence. Studies on DNA binding and transcriptional activities of Xin are also proposed. Relationship of Xin to other genes in the controlling pathway for looping will be studied in Nkx2.5-, MEF2C-, or dHAND-knockout mice. Aim 3 is to perform assays to assess roles of DEGs in cardiac morphogenesis, including antisense studies, epithelial-mesenchymal cell transformation, and knockout and transgenic mice.